Increased matriptase zymogen activation in inflammatory skin disorders

Abstract

Matriptase, a type 2 transmembrane serine protease, and its inhibitor hepatocyte growth factor activator inhibitor (HAI)-1 are required for normal epidermal barrier function, and matriptase activity is tightly regulated during this process. We therefore hypothesized that this protease system might be deregulated in skin disease. To test this, we examined the level and activation state of matriptase in examples of 23 human skin disorders. We first examined matriptase and HAI-1 protein distribution in normal epidermis. Matriptase was detected at high levels at cell-cell junctions in the basal layer and spinous layers but was present at minimal levels in the granular layer. HAI-1 was distributed in a similar pattern, except that high-level expression was retained in the granular layer. This pattern of expression was retained in most skin disorders. We next examined the distribution of activated matriptase. Although activated matriptase is not detected in normal epidermis, a dramatic increase is seen in keratinocytes at the site of inflammation in 16 different skin diseases. To gain further evidence that activation is associated with inflammatory stimuli, we challenged HaCaT cells with acidic pH or H(2)O(2) and observed matriptase activation. These findings suggest that inflammation-associated reactive oxygen species and tissue acidity may enhance matriptase activation in some skin diseases.

Fig. 1.

Lack of matriptase activation in normal epidermis. Samples of normal human skin were used to prepare frozen sections that were stained with hematoxylin and eosin (H & E; A) or immunostained with monoclonal antibodies to detect total matriptase M32 (total MTP; B), total hepatocyte growth factor activator inhibitor (HAI)-1 M19 (HAI-1; C), or activated matriptase M69 (activated MTP; D). Mouse IgG was used as the negative control (not shown). SB, stratum basale; SS, stratum spinosum; SG, stratum granulosum; SC, stratum corneum. Staining intensity is expressed as ++ for strong positive, + for positive, +/− for weak positive, and − for negative. Bars = 25 μm.

Fig. 2.

Expression and subcellular localization of matriptase and HAI-1 in human oral mucosa. Sections of paraffin-embedded human oral mucosal tissue were stained by immunohistochemistry for total matriptase with the MAb M24 (A) and for HAI-1 with MAb M19 (B), as indicated. The micrographs are also presented at higher magnification (C and D) for the subcellular localizations. Bars = 50 μm (A and B), 10 μm (C and D). SS-SL, stratum spinosum-surface layer.

Fig. 3.

Increased matriptase activation in ruptured epidermal cyst. Tissue sections of frozen human epidermal cyst specimens were analyzed by immunohistochemistry for morphology by H & E staining (A) and for total matriptase (C), activated matriptase (D), HAI-1 (E), and negative control using mouse IgG (B). Bars = 50 μm.

Fig. 4.

Increased matriptase activation in chronic eczema. Tissue sections of frozen human skin specimens from chronic eczema patients were analyzed by immunohistochemistry for morphology by H & E staining (A) and for total matriptase (C), activated matriptase (D), HAI-1 (E), and negative control using mouse IgG (B). Bars = 50 μm.

Fig. 5.

Increased matriptase activation in inflamed intradermal nevus, prurigo nodularis, and natural killer cell lymphoma with skin metastasis. Tissue sections of frozen human skin specimens from 3 skin disorders, including intradermal nevus (A and B), prurigo nodularis (C and D), and natural killer cell lymphoma with skin metastasis (E and F), were analyzed by immunohistochemistry for morphology by H & E staining (A, C, E) and for activated matriptase (B, D, F). Bars = 50 μm.

Fig. 6.

Human keratinocyte HaCaT cells activate matriptase in response to extracellular acidity. Immortalized human keratinocyte HaCaT cells were exposed to a pH 6.0 buffer or basal medium as negative control for 30 min. Cell lysates were prepared and analyzed by immunoblot analysis for total matriptase (total MTP), HAI-1, and activated matriptase (activated MTP). Matriptase and prostasin were robustly activated and rapidly inhibited by HAI-1 to form a 120-kDa complex and an 85-kDa complex, respectively. Both complexes were clearly detected by HAI-1 MAb.

Fig. 7.

H₂O₂ exposure induces matriptase activation in HaCaT cells. HaCaT cells were exposed to increasing concentrations of H₂O₂ for 6 h (A) or to 500 μM H₂O₂ for the indicated times (B). Cell lysates were prepared and analyzed by immunoblot analyses for total matriptase (total MTP), activated matriptase (activated MTP), and HAI-1.

Fig. 8.

Schematic illustration of the regulation of matriptase-driven serine protease network by inflammation in skin diseases. A matriptase-driven serine protease network, comprised of matriptase as the initiator protease, prostasin, a downstream protease substrate of matriptase, and HAI-1, a Kunitz-type serine protease inhibitor, is expressed at high levels at the cell surface of living keratinocytes in normal skin, where the activation of the system is tightly regulated. In some skin disease with inflammation, proinflammatory cells are recruited that release reactive oxygen species (ROS) and increase tissue acidity. Both the ROS and acidity stimulate nearby keratinocytes to activate matriptase. The active matriptase rapidly activates prostasin before the inhibitor HAI-1 forms complexes with the active matriptase and active prostasin.